The use of administrable conjugates systems in which biomolecules (II) targeted to specific sites or receptors in the organism of patients are coupled to diagnostically active ligands (I) acting as signal generator means to provide diagnostically useful information has become widely known in recent years.
The diagnostically active agents may include materials capable of generating a response signal to be picked-up by suitable receiving instruments, electronically processed and converted to display. For instance, an electromagnetic signal can be detected and processed by appropriate equipment, and finally converted to data medically interpretable by suitably trained personnel. Among diagnostically active agents, one may cite radionuclides (.beta.- or .gamma.-emitters) detectable by counting and scintillation, iodinated X-ray opacifiers which provide a contrast effect in radio-investigations, magnetically responsive materials (ferromagnetic, paramagnetic and super-paramagnetic substances) which provide contrast effects in magnetic resonance imaging (MRI), and microbubbles as well as microballoons which provide contrast effects in ultrasonic echographic imaging.
The coupling in the foregoing conjugates generally relies on the assistance of a bond or a linker (L) having chemically reactive functions designed to effect covalent bonding between moieties (I) and (II). A number of systems which can be presented by the general formula (I-L-II) i.e. systems comprising moieties (I) and (II) connected chemically by a linker (L) are known in the art.
Many different proposal known include for instance, polymeric microspheres containing magnetically active Fe.sub.3 O.sub.4 particles in which mainly acrylics polymers containing reactive functions, for instance polyacrylamide, polyacrylic acid and the like, are tagged with covalently bonded fluorescent dyes, lectins, antigens, antibodies and other biologically active substances, thus allowing detection and localization of specific carbohydrates residues, hormone receptors and other specific cell surface components.
Superparamagnetic metal oxide particles usable in-vivo for the diagnostic localization of cells or tissues recognized by a particular bioaffinity adsorbent coupled to the particles and also for magnetically directing therapeutically active agents coupled to the particles to pathological sites have also been proposed. The magnetic particles attached to a silane linker by a silanization reaction involving reactive silanes R--Si(OX).sub.3 in which X is a lower alkyl and R represents any aliphatic or aromatic sequence terminating in --NH.sub.2, --OH, --SH, aliphatic hydrophobic, or amphipatic function are reported to be suitable for covalently coupling to a bioaffinity absorbent.
Similarly, administrable magnetic microparticles (for instance magnetite) coupled to substances having binding affinity for organic tissues have also been known. Thus for example, tissue-specific substances such as antibodies, neurotransmitters, hormones, metabolites, enzymes, toxins, and natural or synthetic drugs have been coupled to magnetite particles by coating the particles biodegradable polymers carrying reactive functional groups and linking to the tissue-specific substances through said reactive groups. The tissue-specific targeted magnetite microparticles administered by injection into the blood stream are thus transported to targeted organs or tissues where they operate as contrast enhancers in MRI investigations of said organs.
Methods and reagents for the in-vivo tagging of polymorphonuclear leukocytes (PMN), e.g. lymphocytes, with a medically useful metal ion, including radioisotopes and paramagnetic elements, and subsequent detection of the PMN trafficking and sites of concentrated leukocytes within the organism by radiodetection or MRI have also been suggested. In the suggested method there is administered an effective amount of a formulation comprising a leukostimulatory reagent (a lectin) bound to a useful metal species under conditions allowing the reagent to attach to leukocytes, whereby the concentrations of the stimulated leukocytes are subsequently ascertained by detection and quantitation of the metal using conventional measuring means.
EP-A-0 398 143 (A. J. Fischman et al.) discloses labeled chemotactic peptides to image focal sites of infection or inflammation. The labeled peptides described in this reference are schematized by the formula EQU N(X)-Y-Leu-Phe-Z-W
where N is nitrogen, X is a protective formyl, acetyl or t-Boc group; Y is methionine or norleucine; W is a label, e.g. an EDTA or DTPA chelate of a radioactive or paramagnetic isotope and Z is a covalent bond or a linker sequence. The reference also describes the injection of the radioactive labeled chemotactic peptides into experimental rats previously infected in the thighs with strains of E. coli, and the subsequent localization of the infection sites by hourly serial .gamma.-camera images.
Moreover, derivatized hydrophilic-hydrophobic bloc copolymers have been disclosed to bridge substrates to targeting proteins. For instance, WO-A95/06251 (University of Utah) discloses derivatized Pluronic.RTM. and Tetronic.RTM. compounds carrying reactive groups at the end of the PEG blocks. The derivatized copolymers are used to coat by adsorption hydrophobic surfaces (e.g. polystyrene beads and similar carriers), which coated surface then function as substrates for immobilizing proteins and alike substances (applications: e.g. ELISA tests).
Although the achievements of the prior art have merit, they may suffer from some inevitable drawbacks related to preparing the systems of the general formula (I-L-II) by conventional chemical means which are tedious and costly.
Another disadvantage of the systems known so far is linked to the problem of metabolisations or elimination from the body of these complex molecules which once served their purpose i.e. imaging of the targeted tissue do not have further purpose or reason d'etre. When such complex systems are used to deliver a therapeutic agent to a given site, the depletion of the therapeutically active substance simultaneously causes at least a partial digestion and susequent elimination of the complex molecule. However, when such systems are used for imaging elimination of the used molecule becomes a problem.
The present invention in which there are also used the linker properties of derivatized hydrophobic-hydrophilic bloc copolymers constitute a serious advance toward simplification of the coupling between the magnetically responsive moieties (I) and the homing proteins or peptides (II).